Requirement of p53 targets in chemosensitization of colonic carcinoma to death ligand therapy

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exhibits specific tumoricidal activity and is under development for cancer therapy. Mismatch-repair-deficient colonic tumors evade TRAIL-induced apoptosis through mutational inactivation of Bax, but chemotherapeutics including Camptosar (CPT-11) restore TRAIL sensitivity. However, the signaling pathways in restoring TRAIL sensitivity remain to be elucidated. Here, we imaged p53 transcriptional activity in Bax-/- carcinomas by using bioluminescence, in vivo, and find that p53 is required for sensitization to TRAIL by CPT-11. Small interfering RNAs directed at proapoptotic p53 targets reveal TRAIL receptor KILLER/DR5 contributes significantly to TRAIL sensitization, whereas Bak plays a minor role. Caspase 8 inhibition protects both CPT-11 pretreated wild-type and Bax-/- HCT116 cells from TRAIL-induced apoptosis, whereas caspase 9 inhibition only rescued the wild-type HCT116 cells from death induced by TRAIL. The results suggest a conversion in the apoptotic mechanism in HCT116 colon carcinoma from a type II pathway involving Bax and the mitochondria to a type I pathway involving efficient extrinsic pathway caspase activation. In contrast to Bax-/- cells, Bak-deficient human cancers undergo apoptosis in response to TRAIL or CPT-11, implying that these proteins have nonoverlapping functions. Our studies elucidate a mechanism for restoration of TRAIL sensitivity in MMR-deficient Bax-/- human cancers through p53-dependent activation of KILLER/DR5 and reconstitution of a type I death pathway. Efforts to identify agents that up-regulate DR5 may be useful in cancer therapies restoring TRAIL sensitivity.

Fig. 1.

Etoposide and CPT-11 sensitize Bax^-/- cells to TRAIL. Bax^-/- HCT116 human colon cancer cells were not treated (NT) or treated for 4 h with 10 ng/ml his-tagged TRAIL (TR), for 16 h with etoposide (ETO) or CPT-11 (CPT), or for 16 h with etoposide or CPT-11 followed by TRAIL for 4 h (ETO+TR or CPT+TR). Cells were harvested after treatment for flow cytometric analysis. (A) Example of flow cytometric analysis for active caspase 3-expressing cells. (B) The percentage of cells expressing activated caspase 3 is shown (10,000 cells were examined for each sample).

Fig. 2.

p53 is required for sensitizing Bax^-/- cells to TRAIL. Bax^-/- HCT116 cells were infected with Ad-E6 or Ad-LacZ for 16 h. The infected cells were treated as described in the Fig. 1 legend. (A) p53 protein was determined by Western blot and active caspase 3 was analyzed by flow cytometry. (B) Infected cells were transfected with PG13-Luc and pRL-SV40 plasmids. Luciferase activity was determined by Fisher luminometer (Lower). PG13-Luc and pRL-SV40 stably transfected Bax^-/- HCT116 cells were treated as described above, images were obtained by using the cooled charge-coupled camera of the in vivo imaging system (Upper).

Fig. 3.

Optical imaging of p53 activity in living mice. PG13-Luc and pRL-SV40 stably transfected Bax^-/- HCT116 cells were infected with Ad-LacZ or Ad-E6 for 16 h. Cells (1 × 10⁶) were s.c. implanted into left or right forearm. CPT11 (80 mg/kg) was administered by i.p. injection. After 0 or 16 h of treatment, coelenterazine was injected 3 min before imaging, 2 h apart, followed by d-luciferin injection (5 min before imaging). A whole body image was acquired by using the cooled charge-coupled device camera. Each image was acquired at the same time, relative to the injected substrate, and all of the images are shown at the same scale.

Fig. 4.

The effects of KILLER/DR5, Bak inhibition, and caspase 8 or 9 inhibition on sensitization of Bax^-/- cells to TRAIL after DNA damage. Bax^-/- HCT116 cells were transfected with KILLER/DR5 siRNA (A) or Bak siRNA (B). LacZ siRNA oligos were used as a control. Twenty-four hours after transfection, cells were treated as described in the legend to Fig. 1. KILLER/DR5 and Bak expression were determined by Northern blots. Cells were harvested, stained with propidium iodide, and analyzed by flow cytometry. The sub-G₁ content of 20,000 cells was examined for each sample. (C) Differential effects of caspase 8 (Z-IETD-FMK) and caspase 9 (Z-LEHD-FMK) inhibitors on TRAIL-induced apoptosis. Wild-type or Bax^-/- HCT116 cells were pretreated with 10 μg/ml CPT-11 for 16 h, after which 20 μM caspase 8 or 9 inhibitor was added; after 2 h, TRAIL (10 ng/ml) plus 1 μg/ml anti-His-6 was added for 4 h before sub-G₁ analysis. Extracts were collected and analyzed by Western blotting for the expression of caspase 9.

Fig. 5.

Susceptibility of human colon cancer cells to multiple apoptotic stimuli. Wild-type (WT) and Bax^-/- HCT116 cells were transfected with Bak siRNA or LacZ control siRNA oligos. Twenty-four hours after transfection, cells (WT, Bak-deficient, Bax^-/-, Bax^-/- Bak-deficient, and p53^-/-) were not treated or treated with 50 μg/ml CPT-11, 0.5 μM staurosporine, or 100 ng/ml TRAIL and 1 μg/ml anti-His-6 antibody for 24 h. Cell death was quantified by flow cytometric detection of propidium iodide staining.